Process for producing edible sterol fatty acid esters

ABSTRACT

According to the present invention, there is provided a process for enzymatically producing dietary sterol fatty acid esters having physiological activities from phytosterols and fatty acids or oils and fats using lipase as a catalyst; the synthetic reaction conditions and the following purification steps are so structured that dietary sterol fatty acid esters superior in sensory qualities including color, odor and taste, and safety, which is applicable as a general food, a health food or pharmaceuticals can be produced.  
     In a process for enzymatically producing physiological active dietary sterol fatty acid esters (step  124 ) from sterols derived from vegetable (step  100 ) and fatty acids (step  102 ) or fats and oils containing triacylglycerol as the main component (step  202 ) employing lipase as a catalyst, according to the present invention, the synthetic reaction of sterol fatty acid esters by the lipase is carried out for a predetermined time period in a system at a predetermined temperature and water content, enzyme inactivation treatment, dehydration treatment and enzyme protein removal treatment are carried out, and unreacted sterols and fatty acids are removed by molecular distillation treatment (step  110 ), coloring components are removed by treatment with an adsorbent (step  116 ), odorous components are removed by steam distillation treatment (step  122 ), and dietary sterol fatty acid esters superior in sensory properties and safety (step  124 ) is obtained.

TECHNICAL FIELD

The present invention relates to a process for enzymatically producing aphysiological active sterol fatty acid esters from phytosterols and freefatty acids using lipase as catalyst, wherein dietary sterol fatty acidesters which are excellent as a food from a view point of sensoryproperties and safety is obtained.

The present invention relates also to a process for producing a dietarysterol fatty acid ester of high quality from phytosterols and fats andoils having triacylglycerol as a main component.

BACKGROUND ART

Sterols such as P-sitosterol, which are obtained as a part ofnon-saponified products in the refining process of vegetable oils suchas soybean oil and rapeseed oil, have been known to have an action forlowering the blood serum cholesterol level. As it has recently becomeevident that β-sitostanol, a saturated form of β-sitosterol, has a morepowerful blood serum cholesterol lowering effect than β-sitosterol, ithas been receiving increasing attention.

However, since the above-mentioned free sterols and free stanols areinsoluble in the micellar phase in a digestive organs, these forms arehardly appropriate for their intake to develop their physiologicaleffects. In order to improve the fat-solubility thereof, it has beenproposed to ingest them in the form of sterol fatty acid esters, andrecently, it has been attempted to add sterol fatty acid esters tovarious food products such as margarine with phytosterols, and some ofthem are commercially available.

By the way, sterol fatty acid esters have hitherto not been used infoods, but used in a cholesteric liquid crystal and as a hydrophilicbase material for pharmaceuticals and cosmetics.

Therefore, the sterol fatty acid esters have been produced by chemicalsynthesis with an acid or base catalyst. In chemical synthesis, however,the reaction is generally conducted under severe conditions; thereforeseveral problems may arise such as degraded quality of a product andgeneration of undesirable by-products. In addition to that, the productmight be contaminated with the by-products and the reaction catalyst. Ithas been unavoidable to employ highly complicated purification stepsafter the synthetic reaction.

In this context, the use of an enzyme such as cholesterol esterase andlipase has been recently studied.

Cholesterol esterase and lipase are both categorized as one ofcarboxylic acid ester hydrolases and cholesterol esterase is defined asan enzyme, which generates a free sterol and a free fatty acid from acholesterol fatty acid ester through hydrolysis.

Lipase (which usually means triacylglycerol lipase) is defined as anenzyme, which can generate glycerol and free fatty acids from glycerolfatty acid esters through hydrolysis.

Incidentally, many enzymes are found that the enzymes have bothcholesterol esterase activity and lipase activity (see D. Lombardo etal.: Biochem. Biophys. Acta., 527, (1978), 142-149, D. Lombardo et al.:Biochem. Biophys. Acta, 611 (1980), 136-146, 147-155), and even now nota few examples are known which cannot be distinctly classified whethercholesterol esterase or lipase.

The above-mentioned enzymes are known to be capable of catalyzing thehydrolysis reaction of carboxylic acid ester in common cases and, on theother hand, also capable of catalyzing the synthetic reaction of ester.

Lawrence A. et al. indicated that sterol ester hydrolase derived fromcanine pancreatic juice, which is known as cholesterol esterase, maycatalyze the synthesis of cholesterol oleic acid esters from freecholesterols and free oleic acids (Biochem. Biophys. Acta., 231 (1971)558-560).

D. Lombardo et al. also indicated that cholesterol esterase derived fromhuman pancreatic juice is capable of catalyzing the synthesis ofcholesterol fatty acid esters (Biochimie et al., 1980, 62, 427-432).

Myojo et al. confirmed that lipase is capable of catalyzing thesynthesis of cholesterol fatty acid esters (JP 5-33712 B1).

As shown above, it has been indicated that cholesterol fatty acid esterscan be synthesized using enzymes as opposed to the afore-mentionedchemical synthesis.

However, all of the conventional production examples mentioned above arereferring only to the synthetic reaction for sterol fatty acid esters asa general chemical product, and are not intended for the production ofsterol fatty acid esters for use as general foods, health foods orpharmaceuticals. In other words, with respect to the synthetic reactionconditions and the subsequent purification process, no consideration isgiven for achieving good sensory qualities including color, odor andtaste, and safety of the sterol fatty acid esters, which are importantfactors for general foods, health foods and pharmaceuticals.

Therefore, it has been difficult to employ the sterol fatty acid estersproduced in the conventional process for foods and the like.

The above-described problem exists not only in the production whereinfree fatty acids is employed as the fatty acids, but also in theproduction wherein fats and oils containing triacylglycerol as the maincomponent are employed as starting materials.

DISCLOSURE OF THE INVENTION

Accordingly, a first object of the present invention is to provide aprocess for enzymatically producing a physiological active dietarysterol fatty acid esters from phytosterol derived from vegetable andfree fatty acids using lipase as a catalyst, in which synthetic reactionconditions and the subsequent purification process are so constructedthat the resulting dietary sterol fatty acid ester is superior insensory properties including color, odor, and taste, and safety, and iscapable of being employed as general foods, health foods orpharmaceuticals.

A second object of the present invention is to provide a process forproducing dietary sterol fatty acid esters which is also superior inqualities as those described above, from phytosterols and fats and oilscontaining triacylglycerol as the main component, as starting materials.

The present invention for achieving the above-mentioned first object ischaracterized by a process for enzymatically producing a physiologicallyactive dietary sterol fatty acid esters from phytosterols and free fattyacids using lipase as a catalyst, in which synthetic reaction conditionsand the subsequent purification process are so constructed andconsideration is given for achieving good sensory qualities includingcolor, odor and taste and also safety, that the resulting dietary sterolfatty acid ester can be employed as general foods, health foods orpharmaceuticals.

That is, phytosterols and free fatty acids are used as startingmaterials, sterol fatty acid esters synthetic reaction is carried outunder strictly controlled reaction conditions using lipase as acatalyst, and purification process in several stages is carried out toassure the superior quality of the product as a food, thereby a dietarysterol fatty acid ester which is expected to have physiologicalactivities is enzymatically produced.

The invention as recited in claim 1 is characterized by a process forenzymatically producing a physiologically active dietary sterol fattyacid esters from phytosterols and fatty acids using lipase as acatalyst, wherein fatty acids obtained by enzymolysis or high pressurecontinuous decomposition process are used as the fatty acid source, andsynthetic reaction of sterol fatty acid esters by lipase is carried outin a system with controlled temperature and water content for apredetermined time period, followed by enzyme deactivation treatment,dehydration treatment and enzyme protein removal treatment, thenunreacted sterols and fatty acids are removed by molecular distillation,coloring components are removed by treatment with an adsorbent, odorouscomponents are removed by steam distillation, thereby a dietary sterolfatty acid ester of high quality from a view point of sensory propertiesand safety is obtained.

The invention as recited in claim 2 depended on claim 1 is characterizedin that water content is controlled to be 50% or less when the syntheticreaction of sterol fatty acid esters is carried out by lipase.

The invention as recited in claim 3 depended on claim 1 is characterizedin that the synthetic reaction of sterol fatty acid esters is carriedout using mesophilic lipase, and in this case, the reaction is carriedout at temperature between 30° C. to 50° C., and completed within 48hours.

The invention as recited in claim 4 depended on claim 1 is characterizedin that the synthetic reaction of sterol fatty acid esters is carriedout using thermostable lipase, and in this case, the reaction is carriedout at temperature between 50° C. and 90° C., and completed within 24hours, besides a substance capable of preventing the inactivation of anenzyme is added when the synthetic reaction of sterol fatty acid estersis carried out by lipase, and further a substance having anti-oxidantaction is added when the synthetic reaction of sterol fatty acid estersis carried out by lipase.

The invention as recited in claim 5 depended on claim 1 is characterizedin that a molecular distillation apparatus is employed for removingunreacted sterols and fatty acids by molecular distillation treatment,and the molecular distillation is carried out under a pressure of 13.3Pa or lower at a temperature of 150 to 250° C., and the moleculardistillation is repeated several times.

The invention as recited in claim 6 depended on claim 1 is characterizedin that coloring components are removed by treatment with activated clayas an adsorbent agent, which is used in an amount of 0.1 to 50 wt %based on the weight of the materials to be treated, in the presence ofan organic solvent such as hexane.

The invention as recited in claim 7 depended on claim 1 is characterizedin that odorous components are removed by steam distillation, in whichthe steam distillation is performed at 1330 Pa or lower and at 100 to150° C. to prevent the production of trans fatty acids.

The invention as recited in claim 8 depended on claim 1 is characterizedin that the sterol fatty acid esters obtained as the end product hassterol fatty acid esters content of 90 wt % or more, the peroxide valueof 15 or lower, the acid value of 3 or lower and a color scale (Gardner)of 6 or lower and is almost odorless as determined by a sensory test.

The invention as recited in claim 9 depended on claim 1 is characterizedin that lipase is added in a step-wise manner.

The invention as recited in claim 10 is characterized in that whensterol fatty acid esters as recited in claim 1 are used for food, it isused in the form where the sterol fatty acid esters are previously mixedwith fats and oils containing triacylglycerol as the main component.

The above-mentioned invention (which corresponds to the first aspect ofthe invention with relation to the after-mentioned first Embodiment)will be explained in connection with FIG. 1.

According to the present invention, sterols used as starting materials(refined sterol, step 100) may be any sterol as far as it isphytosterols derived from vegetables such as soybean and rapeseed, andexamples thereof include β-sitosterol, campesterol, brassicasterol,stigmasterol and choresterol. In addition to that, stanols such asβ-sitostanol, a saturated form of β-sitosterol, can be employed as well.These sterols and stanols may be in the form of free sterols and freestanols or in the form of an ester in which they are bonded with othersubstances.

As for fatty acids (free fatty acid, step 102), any saturated fatty acidor unsaturated fatty acid having one or plural double bonds, having 4 to32 carbon atoms and derived from an animal or a vegetable, can be used.Examples thereof include myristic acid, palmitic acid, stearic acid,oleic acid, linoleic acid, α-linolenic acid, γ-linolenic acid,arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid, anddocosahexaenoic acid. Typically straight-chain fatty acids are employedin the most cases, but branched-chain fatty acids can be employed aswell.

According to the present invention, it is recommendable to employ fattyacids, which are obtained by enzymatic decomposition or continuous highpressure decomposition process, to obtain sterol fatty acid esterssuitable for a food.

Next, the synthetic reaction of sterol fatty acid esters (step 104) andthe resulting product thereof (step 106) will be explained.

In the present invention, the lipase to be used as a catalyst for thesynthetic reaction of sterol fatty acid esters may be any lipase derivedfrom various microorganisms, animals and plants. The lipase derived froma microorganism include, for example, those derived from microorganismsof the genera Candida, Alcaligenes, Mucor, Rhizopus, Pseudomonas andGeotricum.

The lipase derived from an animal include, for example, those originatedfrom porcine pancreas. It is preferable to use lipase derived fromCandida cylindracea. When the synthetic reaction is performed under hightemperature conditions, thermostable lipase may be used as well.

The enzyme may be used in the purified or partially purified form. Inthe case where lipase derived from microorganism is employed, either themicroorganism cell bodies or a culture of the microorganism may be used.The above-mentioned enzyme may also be in the free state or beimmobilized onto any of various supports such as cerite.

The enzymes used in the present invention may be any one which cancatalyze the same reactions as those catalyzed by lipase. For example,cholesterol esterase is known to be capable of catalyzing the samereactions as those catalyzed by lipase, and therefore can also be usedin the present invention.

The conditions employed for the synthetic reaction of the sterol fattyacid esters with lipase should be controlled strictly so that sterolfatty acid esters having qualities not only suitable for a food such assensory properties including color, odor and taste, but also assuringsafety can be produced at a low cost. Hereinafter the conditions forsynthetic reaction in step 104 will be explained.

The amount of the enzyme used may be 50,000 units or less, preferably10,000 units or less, per gram of the starting material sterols. (Oneunit is defined as an amount of the enzyme capable of releasing 1 μmoleof a fatty acid from olive oil in 1 minute.) To avoid the deteriorationdue to heat treatment in the production process and to produce a moreinexpensive end product, it is desirable to use the enzyme in a smallestpossible amount, preferably in an amount of 1,000 units or less per gramof the starting material sterol. In addition to that, the enzyme canalso be added in a step-wise manner during the synthetic reaction toreduce the amount of the enzyme used.

The starting material sterols (step 100) and fatty acids (step 102) canbe used at any ratio, however, it is preferable to have a higher ratioof fatty acids to sterols in order to improve the synthetic ratio, theratio of fatty acids to sterols is preferably 50% or higher. As sterolsare solid at a normal temperature, a desirable weight ratio of fattyacids to sterols is 100% or higher in order to further improve theworkability during the production process. Furthermore, as startingmaterial costs and production costs should be reduced for obtaining amore inexpensive product, the use ratio of fatty acids to sterols of 100to 500% may be preferred.

The sterols and fatty acids, as starting materials, can be directlymixed and supplied to the synthetic reaction, however, a small amount ofwater may be added in order to raise the synthetic ratio. On the otherhand, as the water added must be removed in the purification processafter the synthesis of the sterol fatty acid esters, the amount of thewater used must be as small as possible, for reducing the productioncost, and is preferably up to 50% based on the weight of the startingmaterial sterols.

To minimize the thermal degradation during the reaction, the reaction ispreferably performed at a lower temperature for a shorter time period,desirably at 30 to 50° C. within 48 hours. When the synthesis isperformed at a low temperature, a lipase which can readily exert itsenzymatic activity at such a low temperature is preferably used.

On the other hand, as sterols, one of the starting materials, have veryhigh melting points, they tend to have poor compatibility with the otherstarting material, fatty acids, and that may lead to low syntheticreaction efficiency of the sterol fatty acid esters by lipase. Toovercome this problem, the synthetic reaction can be performed at highertemperature, preferably at 50 to 90° C. using thermostable lipase. Inthis case, however, thermal deterioration of sterol fatty acid estersmay proceed more aggressively or lipase may be inactivated during thereaction, therefore, a substance having anti-oxidant effect such asVitamin E and tea polyphenol may be added to prevent the thermal oroxidative deterioration of the sterol fatty acid esters, and a substancecapable of preventing the inactivation of an enzyme, including a saltsuch as bile salts, a carbohydrate and a protein may be added to preventthe inactivation of the enzyme.

To enhance the efficiency of the synthetic reaction, the reaction isusually carried out with stirring, but in some cases, it can be carriedout in a static state. When the reaction is performed in a static state,an emulsifying agent or the like may be added. An organic solvent suchas hexane may also be used to enhance the efficiency of the reaction,but in this case, the solvent must be removed, which may increase theproduction cost.

The purification of sterol fatty acid esters will be illustratedhereinbelow.

In the present invention, in order to achieve inexpensive production ofsterol fatty acid esters which are superior in qualities suitable for afood including color, odor and taste, also in safety, theabove-mentioned purification process after the synthetic reaction mustbe performed carefully.

At first, after the synthetic reaction of the sterol fatty acid estersis completed, inactivation of the enzyme, dehydration and removal of theenzyme protein are performed. The inactivation of the enzyme is achievedby stirring at 60° C. or higher for about 30 to 120 min. The dehydrationis performed by treatment at 60° C. or higher for a predetermined timeperiod under reduced pressure. The removal of the enzyme protein can beachieved by filtration using a conventional filter paper, filter clothor a filtration filter. If the removal of the enzyme protein by thefiltration is insufficient, then deterioration in quality such ascoloration may be caused by the heating in subsequent processes.Therefore, the enzyme protein must be removed completely. For moreeffective removal, a filter aid such as diatomaceous earth can bepreviously added, and then stirred and filtered. So far, we haveexplained the treatment up to step 106.

After the removal of the enzyme protein (step 106), the sterol fattyacid esters still contain unreacted sterols, fatty acids, coloringcomponents, and odorous components.

Therefore, according to the present invention, molecular distillation(step 110) is next performed to remove the sterols and fatty acids (step108) efficiently.

During this process, the end product, the sterol fatty acid ester, isobtained as the residue (step 112) and unreacted sterols and fattyacids, and a part of coloring components and odorous components areobtained as a fraction (step 108).

As the apparatus for the molecular distillation, any type can be usedwhich is selected from falling film type, centrifugal type or any othertype of short pass distillation apparatus. The molecular distillation ispreferably performed at 133 Pa or lower and at 100 to 300° C., morepreferably at 13.3 Pa or lower and at 150 to 250° C. The moleculardistillation may be performed repeatedly several times.

Subsequently, treatment with an adsorbent is performed to removecoloring components and the like. This is because sterol fatty acidesters (residue) after the molecular distillation contains coloringcomponents derived from the starting materials, coloring componentsresulting from the heating during the distillation, and odorouscomponents and the like (step 112).

To remove the coloring components (step 114) efficiently according tothe present invention, sterol fatty acid esters are treated with anadsorbent (step 116). The adsorbent used in this case includes thosetypically used for purification of fats and oils, such as activatedclay, acidic clay, activated carbon, silica, silica-magnesia and so on,but activated clay is preferred. The adsorbent is preferably added in anamount of 0.1 to 50 wt %, more preferably 1 to 20 wt %, based on theweight of the starting material to be treated. For more efficientdecoloring, it is preferred to employ activated carbon in a non-polarsolvent such as hexane. The solvent is preferably used in an amount of0.1 to 50 times, more preferably 0.5 to 20 times that of the startingmaterial to be treated. When an organic solvent is employed, the solventmust be removed after the treatment with the adsorbent. The treatmentwith the adsorbent can be repeated for several times.

As the final step, steam distillation (step 122) is performed to removeodorous components (step 120) and the like. Odorous components (step120) derived from the starting materials or generated in the precedingsteps must be removed from sterol fatty acid esters obtained after thedecoloring (step 118), in order for sterol fatty acid esters to be usedas a food. When the decoloring is performed using an organic solvent,the organic solvent may remain in the product even after the solventremoval treatment, therefore, such organic solvent must be removedcompletely. Steam distillation treatment (step 122) makes it possible toremove the above-mentioned odorous components (step 120) and theremaining organic solvent. In the steam distillation, any type ofapparatus may be used, including those of continuous type,semi-continuous type and batch type. It is desirable to perform thesteam distillation under the conditions of 13.3 kPa or lower and 50 to250° C., preferably 1330 Pa or lower and 100 to 150° C. By lowering thetemperature, the production of trans fatty acids can be prevented. Thesteam distillation may be repeated several times.

The obtained sterol fatty acid esters (step 124) can be used as a foodas it is; however, because of extremely poor fluidity and workability,sterol fatty acid esters are preferably used in the form where it ispreviously mixed with other fats and oils, such as those containingtriacylglycerol as the main component. In this case, any mixing ratiomay be employed depending on the intended use, but it is desirable tomix the fat and oil in an amount of 30 to 300 wt % based on the weightof sterol fatty acid esters in order to improve the workability. As thefats and oils containing triacylglycerol as the main component, foodfats and oils, such as soybean oil and rapeseed oil may be used. Insteadof the fats and oils containing triacylglycerol as the main component,other fats and oils containing other components such as diacylglycerolmay also be used.

The sterol fatty acid esters obtained as the end product according tothe present invention, is almost tasteless, odorless and pale yellow incolor and is superior in safety, and therefore has qualities suitablefor a general food, a health food and pharmaceuticals. The sterol fattyacid esters have the potential effect of reducing cholesterol level.Accordingly, the sterol fatty acid esters are expected to be used, notonly as a functional material, in a general food product includingmargarine and dressing, but also in a health food product, or in apharmaceuticals and the like.

As described heretofore, according to the present invention, a syntheticreaction of sterol fatty acid esters is carried out under strictlycontrolled reaction conditions, employing phytosterols and fatty acidsas starting materials, and employing lipase as a catalyst, and carryingout purification treatment in several steps following the synthesis, toobtain a quality product for use as a food, thereby providing specificadvantages of the present invention, that is dietary sterol fatty acidesters of high physiological activities and high quality can beproduced.

In contrast to the present invention, prior art (for example JP 5-33712B1) describes production of sterol fatty acid esters by use of enzymaticactivity as well.

However, the production process described in prior art is not aiming atproduction of the dietary sterol fatty acid ester.

Therefore, there are the following differences between the productionprocess described in prior art and the present invention.

(a) In the prior art, it is described that fatty acids having 2 to 32carbon atoms can be used as the starting material fatty acids, butaccording to the present invention, the starting materials are thoseobtained by enzymolysis or continuous high pressure decompositionprocess.

Therefore, according to the present invention, there is the advantagethat the deterioration of the starting material fatty acids is littleand a product of high safety as a food can be produced.

(b) In the prior art, it is described that lipase or cholesterolesterase can be employed, while in the present invention, the enzyme isadded in a step-wise manner.

Therefore, the deactivation of the enzyme during the reaction processcan be reduced in the present invention and the amount of the enzymeused can be decreased. Therefore there is another advantage that theproduct can be obtained at low cost.

(c) According to the present invention, a substance which inhibits theinactivation of an enzyme is added or a substance which has anti-oxidantaction is added while the synthetic reaction of the sterol fatty acidesters is carried out by lipase; however, there is no such descriptionin the prior art.

(d) In the prior art, extraction with an ether, or column chromatographyprocess are described for removal of unreacted free fatty acids or freesterols; however, ethers cannot be used for production of a food, andthe column chromatography process increases the cost.

On the contrary, in the present invention, unreacted free fatty acidsand free sterols are removed by molecular distillation. Therefore, thereis the advantage that products can be obtained safely at a low cost.

(e) In the prior art there is no description of construction forremoving coloring components and odorous components. Therefore, theresulting product cannot be used for a food.

On the contrary, in the present invention, treatment with an adsorbentis carried out for removal of coloring components, and steamdistillation is carried out for removal of odorous components.Therefore, it has the advantage that a food which is superior in sensoryproperties including color, odor and taste, and safety can be produced.

As described above, when the present invention is compared with theprior art, the present invention has advantages specific to the presentinvention, that sterol fatty acid esters for a food can be producedsafely and at a low cost.

Next, the second aspect of the present invention in order to achieve theabove-mentioned second object will be explained in connection with FIG.2.

The present invention to achieve the above-mentioned second object ischaracterized in that quality dietary sterol fatty acid esters areproduced from sterols derived from vegetable and a fat and oilcontaining triacylglycerol as the main component as starting materials.

That is a synthetic reaction of sterol fatty acid esters is carried outunder strictly controlled reaction conditions, by employing phytosterolsand fats and oils containing triacylglycerol as the main component asstarting materials, and by employing an enzyme having a lipolyticactivity, as a catalyst, and by carrying out purification treatment inseveral steps following the synthesis, to obtain a dietary sterol fattyacid ester of high quality enzymatically.

The invention recited in claim 11 according to the present invention ischaracterized by a process for producing dietary sterol fatty acidesters comprising;

-   -   carrying out a synthetic reaction of sterol fatty acid esters by        employing phytosterols and a fat and oil containing        triacylglycerols as the main component, as starting material,        and by employing an enzyme having a lipolytic activity in a        system with controlled temperature and water content for a        predetermined time period, then    -   performing enzyme deactivation treatment, dehydration treatment        and enzyme protein removal treatment,    -   performing as a first purification step, molecular distillation        to primarily remove unreacted sterols and fatty acids;    -   performing as a second purification step, treatment with an        adsorbent to primarily remove coloring components; and    -   performing as a third purification step, steam distillation to        primarily remove odorous components,    -   to obtain sterol fatty acid esters being superior in safety and        sensory properties for food.

The invention recited in claim 12 depended on claim 11 is characterizedin that phytosterols composition used as the starting material containsβ-sitosterol in an amount of 20 to 80% by weight.

The invention recited in claim 13 depended on claim 11 is characterizedin that the fat and oil containing triacylglycerol as the main componentwhich is used as the starting material is any one of oils selected fromsoybean oil, rapeseed oil, and olive oil, and used alone or in admixtureor two or more kinds.

The invention recited in claim 14 depended on claim 11 is characterizedin that an enzyme having a decomposition activity of sterol fatty acidesters is employed as the enzyme having a lipolytic activity.

The invention recited in claim 15 depended on claim 11 is characterizedin that an enzyme having a decomposition activity of triacylglycerol isemployed as the enzyme having a lipolytic activity.

The invention recited in claim 16 depended on claim 14 or claim 15, ischaracterized in that the enzyme having an activity of decomposingtriacylglycerol is cholesterol esterase or lipase.

The invention recited in claim 17 depended on claim 11 is characterizedin that the synthetic reaction of sterol fatty acid esters by an enzymehaving a lipolytic activity is carried out for a predetermined timeperiod, in a system with controlled temperature and water content, inwhich the reaction is carried out under conditions of water content of0.1 to 50% based on the weight of the starting materials, at 30 to 60°C., and within 48 hours.

The invention recited in claim 18 depended on claim 11 is characterizedin that the synthetic reaction of sterol fatty acid esters by an enzymehaving a lipolytic activity is carried out for a predetermined timeperiod, in a system with controlled temperature and water content,wherein a thermostable lipolytic enzyme is employed and the reaction iscarried out in the presence of a substance which prevents inactivationof the enzyme and a substance which has an anti-oxidant action, underconditions of the water content of 0.1 to 50% by weight based on theweight of the starting materials, at 50 to 90° C., and within 48 hours.

The invention recited in claim 19 depended on claim 18 is characterizedin that a lipolytic enzyme originated from a microorganism of the genusRhizopus is employed as the thermostable lipolytic enzyme.

The invention recited in claim 20 depended on claim 11 is characterizedin that the molecular distillation treatment, as the first purificationstep, is carried out at 13.3 Pa or lower and at 100 to 250° C. forremoving primarily unreacted sterols and fatty acids.

The invention recited in claim 21 depended on claim 11 is characterizedin that the treatment with an adsorbent for removing primarily coloringcomponents, as the second purification step, is carried out by usingactivated clay in an amount of 0.1 to 50% based on the weight of thematerials to be treated and at 100° C. or lower.

The invention recited in claim 22 depended on claim 11 is characterizedin that the steam distillation treatment, as the third purificationstep, is carried out at 1330 Pa or lower and 50 to 150° C. to removeodorous components, at the same time to prevent the generation of transfatty acids.

The invention recited in claim 23 depended on claim 11 is characterizedin that the end product sterol fatty acid ester has a sterol fatty acidester content of 90 wt % or more, a peroxide value of 15 or lower, anacid value of 3 or lower and a color scale (Gardner) of 6 or lower andis almost odorless as determined by a sensory test.

Then the above-mentioned invention (which corresponds to the secondaspect of the invention with relation to the after-mentioned secondEmbodiment) will be explained in connection with FIG. 2.

Phytosterols (purified sterol, step 200), one of the starting materialsaccording to the present invention, include any sterols which is derivedfrom a vegetable such as soybean and rapeseed, and examples thereofinclude β-sitosterol, campesterol, brassicasterol, stigmasterol, andcholesterol, and stanols such as β-sitostanol, a saturated form thereofcan be employed as well. These sterols and stanols can be used in freeform or in ester form in which they are bonded to other substances.

The other starting material, i.e. the fats and oils containingtriacylglycerol as the main component (triacylglycerol, step 202)includes vegetable-derived oils such as soybean oil, rapeseed oil, oliveoil, palm oil, sunflower seed oil, safflower oil, corn oil, cotton seedoil, sesame oil, rice bran oil, coconut oil, and peanut oil, and theseoils may be used singly or in admixture of two or more kinds. The fatsand oils may be added in an amount of 50 to 500 wt %, preferably 100 to300 wt %, based on the weight of the sterol-containing fraction.

Then the synthetic reaction of the sterol fatty acid esters in thepresent embodiment and the resulting product will be explained inconnection with steps 204 and 206.

The lipolytic enzyme to be used as a catalyst for the synthetic reactionof the sterol fatty acid esters (step 204) may be any of thoseoriginated from various microorganisms, animals and plants. The enzymehaving a lipolytic activity originated from a microorganism include, forexample, those from microorganisms of the genera Pseudomonas,Alcaligenes, Candida, Mucor, Rhizopus and Geotricum. The enzyme having alipolytic activity originated from an animal include, for example, thoseoriginated from porcine pancreas.

The enzyme used in the present invention is preferably one capable ofdegrading sterol fatty acid esters or one capable of degradingtriacylglycerol, and concrete example thereof includes cholesterolesterase and lipase. The former enzyme includes those originated from amicroorganism of genus Pseudomonas, and the latter enzyme includes thoseoriginated from a microorganism of genera Alcaligenes and Candida.

A number of enzymes having both an activity of degrading sterol fattyacid esters and an activity of degrading triacylglycerol have been alsoknown, and according to the present invention, any lipolytic enzymewhich is able to catalyze the synthetic reaction of sterol fatty acidesters can be used without concern for enzyme-classificatory constrains.

When the synthetic reaction of sterol fatty acid esters is performedunder high temperature conditions, a thermostable lipolytic enzyme maybe used. As the thermostable lipolytic enzyme, those originated from amicroorganism of genera Rhizopus is preferred. The enzyme may be used inthe purified or partially purified form. When a lipolytic enzymeoriginated from a microorganism is employed, either the microorganismcell bodies themselves or the culture of the microorganism may be used.The enzyme may also be in the free state or be immobilized onto any ofvarious supports such as cerite.

The conditions to be employed for the synthetic reaction of the sterolfatty acid esters with a lipolytic enzyme according to the presentinvention, should be controlled strictly so that sterol fatty acidesters having qualities not only suitable for a food, such as sensoryproperties including color, odor and taste, but also assuring safety,can be produced at a low cost. The synthetic reaction conditions forstep 204 will be described in the following.

The enzyme may be used in an amount of 50,000 units or less, preferably10,000 units or less per gram of the starting material sterols. (Oneunit is defined as an amount of the enzyme capable of releasing 1 μmoleof a fatty acid from olive oil in 1 minute.) To avoid the deteriorationof the enzyme caused by the treatment under heating in the productionprocess and to produce the end product at lower cost, it is desirable touse the enzyme in a smallest possible amount, preferably in an amount of1,000 units or less per gram of the starting material sterols. Theenzyme can also be added in a step-wise manner during the syntheticreaction to reduce the amount of the enzyme.

In the present invention, to prevent triacylglycerol, diacylglycerol ormonoacylglycerol from remaining after the synthetic reaction of thesterol fatty acid esters, it is preferred to perform the enzymaticreaction by previously adding water in an amount of 0.1 wt % or more. Byperforming the enzymatic reaction in the presence of water of 0.1 wt %or more, triacylglycerol, and coexisting trace amount of diacylglyceroland monoacylglycerol and the like are subjected to hydrolysis reactionand decomposed into free fatty acids and glycerol, and these fatty acidsgenerated by the decomposition also become substrates for the estersynthetic reaction of the sterol fatty acid esters. The efficiency ofthe synthetic reaction can be enhanced, by increasing the amount of thewater added. On the other hand, since the added water must be removed inthe purification process after the synthesis of the sterol fatty acidesters, the amount of water should be minimized for lowering theproduction cost, and a desirable amount is 300% or less, preferably 50%or less, based on the weight of the starting materials.

To minimize the thermal degradation of the product during the syntheticreaction, the reaction is preferably performed at lower temperature forshorter time period, usually at 30 to 50° C. within 48 hours. When thesynthesis is performed at lower temperature, lipolytic enzyme which canreadily exert its enzymatic activity at such lower temperature ispreferably used.

On the other hand, sterols, constituting one main starting material,have very high melting points thereby their compatibility with the othersubstrate, i.e. fats and oils containing triacylglycerol as the maincomponent, is extremely poor, and in some cases, the efficiency of thesynthetic reaction of the sterol fatty acid esters by the lipolyticenzyme becomes low. To overcome this problem, the synthetic reaction canbe performed at higher temperature, preferably at 50 to 80° C. within 24hours using thermostable lipolytic enzyme. In this case, however,thermal deterioration may proceed more aggressively or the enzyme may beinactivated during the reaction, a substance having anti-oxidant effect,such as Vitamin E and tea polyphenol, may be added to prevent thethermal or oxidative deterioration of the sterol fatty acid esters, anda substance capable of inhibiting the inactivation of the enzyme, suchas a salt including a bile salt, a carbohydrate and a protein, may beadded to prevent the inactivation of the enzyme.

To enhance the efficiency of the synthetic reaction, the presentreaction is usually performed while stirring, but in some cases it maybe performed in a static state. When the reaction is performed in astatic state, an emulsifying agent or the like may be added. An organicsolvent such as hexane may also be used to enhance the efficiency of thereaction, but in this case, the solvent must be removed, and that mayincrease the production cost.

After the synthetic reaction of the sterol fatty acid esters iscompleted, inactivation of the enzyme, dehydration and removal of theenzyme protein are performed. The inactivation of the enzyme is achievedby stirring at 60 to 100° C. for about 30 to 120 min. The dehydrationtreatment is performed at 60 to 120° C. for a predetermined time periodunder reduced pressure. The removal of the enzyme protein can beachieved by filtration using a conventional filter paper, filter clothor a filtration filter; however, in case the removal of the enzymeprotein is insufficient, deterioration of the product in quality such ascoloration may likely be caused by the heating in subsequent processes,therefore, the enzyme protein must be removed completely. For moreeffective removal, a filter aid such as diatomaceous earth and clay canbe previously added and then stirred and filtered. So far is thetreatment up to step 206.

Next, the purification process for the sterol fatty acid esters will beexplained.

According to the present invention, in order to produce sterol fattyacid esters having qualities suitable for food including color, odor andtaste, and is superior in safety at low cost, the above-mentionedpurification of the product after the synthetic reaction should beperformed carefully.

As the sterol fatty acid esters obtained after the enzyme proteinremoval treatment (step 206) according to the present invention,contains unreacted sterols and fatty acids, molecular distillation, as afirst purification step, is performed (step 210) to efficiently removethese substances, wherein the sterol fatty acid esters, as the endproduct, is obtained as a remaining fraction (Step 212), while unreactedsterols and fatty acids and parts of odorous components are removed asdistillate fractions (Step 208). Examples of the apparatus for themolecular distillation include those of falling film type, centrifugaltype or any other type of short pass distillation apparatus, but anyapparatus may be used as long as it can achieve the desired vacuumpressure and temperature and can remove the desired free sterols, freefatty acids and other trace components. The molecular distillation ispreferably performed at 133 Pa or lower and at 100 to 300° C., morepreferably at 13.3 Pa or lower and at 100 to 250° C. The moleculardistillation may be performed repeatedly several times. As this processallows the removal of odorous components, which cannot be completelyremoved in the steam distillation (Step 222), the third purificationstep, molecular distillation treatment (step 210) is required to beperformed prior to the steam distillation (Step 222) to produce an endproduct from which odorous components are efficiently removed.

Subsequently, in a second purification step, coloring components (Step214) are primarily removed. The sterol fatty acid esters after themolecular distillation (Step 212) contains coloring components derivedfrom the starting materials and coloring components resulting from theheating during the molecular distillation (step 214), as well as odorouscomponents (step 220). According to the present invention, to remove thecoloring components efficiently, treatment with an adsorbent (Step 216)is performed.

The adsorbent used in this case includes those adsorbents conventionallyused for purification of fats and oils, such as activated clay, acidicclay, activated carbon, silica, silica-magnesia and so on, and the useof activated clay, activated carbon or silica is preferred. These may beused singly or in admixture of two or more kinds. The adsorbent ispreferably added in an amount of 0.1 to 50 wt %, more preferably 1 to 20wt %, based on the weight of the material to be treated. For moreefficient decoloring, the treatment can be carried out with theabove-mentioned adsorbent in a non-polar solvent such as hexane. Thesolvent is preferably used in an amount of 0.1 to 50 times, morepreferably 0.5 to 20 times (by weight), that of the material to betreated. When a non-polar solvent is not used, the decoloring isperformed by adding the adsorbent followed by stirring at 40 to 150° C.for a predetermined time period. This procedure may be performed undernormal atmospheric pressure, but the procedure is preferably performedunder reduced pressure to prevent the material from deterioration and tocarry out more efficient decoloring. A lower pressure is preferred, suchas 13.3 kPa or lower. After the treatment, the adsorbent is removed byfiltration using any conventional filtrating means, such as a filterpaper, filter cloth or a filtration filter. For more effective removal,a filter aid such as diatomaceous earth can be added prior to thefiltration and then stirred and filtered. In the case where a non-polarsolvent is used, it is preferred to dissolve the material to be treatedin the non-polar solvent previously, and then add the adsorbent theretoand stir the resulting reaction mixture at 0 to 60° C. for apredetermined time period.

The adsorbent is removed in the same manner as stated above, and thenthe non-polar solvent is removed by distillation (step 222). When themore complete removal of the coloring components is required or thematerial to be discolored has darker color, it is preferred to repeatthe treatment with the adsorbent several times. When the treatment isrepeated, additional any adsorbent may be added after the filtration ofthe preceding adsorbent and then another round of procedure is performedin the same manner. When a non-polar solvent is used, additional anyadsorbent may be added after the proceeding round of addition of anadsorbent, stirring and filtration without the need of removal of thesolvent, and then the subsequent round is performed. The solvent isremoved after filtration in the final round is completed.

The material may be treated with an acid or alkali prior the treatmentwith the adsorbent, thereby achieving the decoloring more effectively.In this case, the material to be treated may be dissolved in a non-polarsolvent such as hexane to make the material in a micellar state, andthen treated with an acid or alkali. As in this step, additional odorouscomponents may be generated or attached to the sterol fatty acid esters,the treatment with the adsorbent (Step 216) is required to be performedafter the molecular distillation (Step 210) and prior to the steamdistillation (Step 222).

Finally as the third purification step, steam distillation (Step 222) isperformed to remove odorous components (Step 220) and the like. For useas a food, the sterol fatty acid esters after the decoloring is requiredto be free from odorous components derived from the starting material orgenerated in the preceding steps. When the decoloring is performed usingan organic solvent, the organic solvent may remain in the product evenafter the removal treatment of the solvent. Therefore, the remainingorganic solvent must be removed completely. According to the steamdistillation, the odorous components and the organic solvent remainingin the product can be almost completely removed.

In the steam distillation, any type of apparatus that is selected fromcontinuous type, semi-continuous type and batch type apparatus can beused. The steam distillation is desirable to be performed under theconditions of 13.3 kPa or lower and 50 to 200° C., preferably 1330 Pa orlower and 50 to 150° C. The steam distillation at lower temperature isadvantageous as it can prevent generation of trans fatty acids. Thesteam distillation may be repeated several times. As stated previously,in order to completely remove the odorous components that cannot beremoved only through the steam distillation, it is required to performboth of the steam distillation and the molecular distillation (Step210). In this case, it is important to perform the moleculardistillation (Step 210) prior to the steam distillation.

The sterol fatty acid esters (Step 224), the end product of the presentinvention, is almost tasteless, odorless and colorless or pale yellow incolor, superior in safety, and has properties suitable for a generalfood, a health food and pharmaceuticals. The sterol fatty acid estersare expected to have the potential effect of reducing cholesterol level,therefore it is expected to be used, as a functional material, ingeneral food products such as margarine and dressing and health foodproducts and, in the future, in pharmaceuticals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram illustrating a process for producingdietary sterol fatty acid esters according to the first embodiment ofthe present invention.

FIG. 2 is a schematic diagram illustrating a process for producingdietary sterol fatty acid esters according to the second embodiment ofthe present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be described more in detail by way of thefollowing examples. The present invention, however, is not limited tothese specific examples.

The first embodiment according to the first aspect of the presentinvention will be described hereinbelow. The first embodimentcorresponds to the invention illustrated in the above-mentioned FIG. 1.

EXAMPLE 1

Soybean-derived sterol (sterol content: 95 wt %) (100 g) and oleic acid(oleic acid content: 99%) (200 g) were mixed at 40° C. and dissolvedthen lipase powder originated from a microorganism of the genus Candida(360,000 units/g) (2.0 g) was further added thereto while stirring thestarting material mixture solution. The synthetic reaction was carriedout at 40° C. for 24 hours while stirring, then the reaction mixture washeated to 80° C. and stirred at that temperature for. 30 min. toinactivate the enzyme. The resulting product was added with diatomaceousearth (1.0 g), stirred, and then filtered to remove the enzyme protein.The resultant product was subjected to molecular distillation using acentrifugal molecular distillation apparatus at a vacuum pressure of1.33 Pa, an evaporating surface temperature of 230° C. and a flow rateof 2.0 L/hr to give a sterol fatty acid ester (141 g) free fromunreacted free sterols and free fatty acids. For the treatment with anadsorbent, the sterol fatty acid esters were dissolved in hexane (theamount of which was 10 times that of the residue), added with 20 wt %(based on the residue) of activated clay, and then stirred at roomtemperature for 30 min. The resulting solution was filtered to removethe activated clay having coloring components adsorbed thereon, and thefiltrate was evaporated using an evaporator to remove the solvent,thereby giving the sterol fatty acid ester without coloring components(122 g). Finally, the sterol fatty acid esters were subjected to steamdistillation using a batch-type steam distillation apparatus under thecondition of vacuum pressure of 500 Pa, a distillation temperature of150° C. and distillation time of 1 hour, thereby giving the sterol fattyacid ester without odorous components (118 g). The obtained sterol fattyacid ester was almost tasteless, odorless and pale yellow in color. Theanalytic results are shown in Table 1.

The sterol fatty acid esters were mixed with soybean oil in an amount of100 wt % based on the weight of the sterol fatty acid esters, and aproduct having good fluidity was obtained.

EXAMPLE 2

The materials used in EXAMPLE 1, i.e. soybean-derived sterol (sterolcontent: 95 wt %) (100 g) and oleic acid (oleic acid content: 99%) (200g) were mixed with 30 g of water and dissolved then lipase powderoriginated from a microorganism of the genus Candida (360,000 units/g)(2.0 g) was further added thereto while stirring the starting materialmixture solution. The synthetic reaction was carried out at 40° C. for24 hours while stirring, then the reaction mixture was heated to 80° C.and stirred at that temperature for 30 min. to inactivate the enzyme.The resulting product was added with diatomaceous earth (1.0 g),stirred, and then filtered to remove the enzyme protein. The product wasdehydrated at 13.3 kPa and 80° C. with stirring for about 1 hour. Thenpurification was carried out in the same manner as in EXAMPLE 1 andsterol fatty acid esters (123 g) which had improved color, odor andtaste was finally obtained. The obtained sterol fatty acid esters werealmost tasteless, odorless and pale yellow in color. The analyticresults are shown in Table 1.

EXAMPLE 3

The starting materials employed in EXAMPLE 1 were mixed and dissolved at60° C. and thermostable lipase powder originated from a microorganism ofthe genus Rhizopus (10,000 units/g) (3.0 g) was added thereto whilestirring. The synthetic reaction was carried out at 60° C. for 8 hourswhile stirring, then further heated to 100° C. and stirred at thattemperature for 30 min. Then inactivation of the enzyme, removal of theenzyme protein were carried out in the same manner as in EXAMPLE 1. Thepurification was carried out in the same manner as in EXAMPLE 1 to givea sterol fatty acid ester (124 g). The obtained sterol fatty acid esterswere almost tasteless, odorless and pale yellow in color. The analyticresults are shown in Table 1.

EXAMPLE 4

To the starting materials employed in EXAMPLE 2 were added Vitamin E inan amount of 0.5% and bile salt of 0.2% based on the weight of thestarting materials and mixed and dissolved at 60° C. and thermostablelipase powder originated from a microorganism of the genus Rhizopus(10,000 units/g) (3.0 g) was added thereto while stirring. The syntheticreaction was carried out at 60° C. for 8 hours while stirring, thenfurther heated to 100° C. and stirred at that temperature for 30 min.Then inactivation of the enzyme, dehydration and removal of the enzymeprotein were carried out in the same manner as in EXAMPLE 1. Thepurification was carried out in the same manner as in EXAMPLE 1 to givesterol fatty acid esters (127 g). The obtained sterol fatty acid esterswere almost tasteless, odorless and pale yellow in color. The analyticresults are shown in Table 1.

COMPARATIVE EXAMPLE 1

The starting materials employed in EXAMPLE 1 were mixed and dissolved at50° C. and lipase powder originated from a microorganism of the genusCandida (360,000 units/g) (2.0 g) was added thereto while stirring. Thesynthetic reaction was carried out at 50° C. for 36 hours whilestirring. Then molecular distillation treatment was carried out using afalling film molecular distillation apparatus followed by treatment withactivated carbon and sterol fatty acid esters (82 g) were obtained. Thesterol fatty acid ester had a characteristic pungent odor and was brownin color. The analytic results are shown in Table 1. TABLE 1 Ex. 1 Ex. 2Ex. 3 Ex. 4 C. Ex. 1 Sterol fatty TLC-FID % 96.1 97.5 98.7 98.9 95.4acid esters content (wt %) Peroxide value meq/kg 4.5 5.1 12.3 6.4 18.2(POV) Acid value mg-KOH/g 0.2 0.3 0.5 0.4 2.6 (AV) Color Gardner 3− 3−4+ 4− 9+ Scale Odor Sensory almost almost almost almost characteristicevaluation odorless odorless odorless odorless pungent odor TasteSensory tasteless tasteless tasteless tasteless bitterness evaluationTrans fatty acid content GCarea % 1.0 1.0 1.2 1.1 5.0

Next, second embodiment according to the second aspect of the presentinvention will be described. The second embodiment corresponds to theabove-mentioned invention illustrated in FIG. 2.

EXAMPLE 5

Phytosterols (β-sitosterol content:42.5%) (50 g) and refined soybean oil(100 g) were mixed and a suspension in water (50 g) of lipolytic enzymepowder originated from a microorganism of the genus Pseudomonas (50,000units/g) (2.0 g) having an activity of decomposing sterol fatty acidesters in water (50 g) was further added thereto, and the syntheticreaction of sterol fatty acid esters was carried out at 40° C. for 24hours with stirring. Then the reaction mixture was heated to 80° C. andstirred at that temperature for 30 min. to inactivate the enzyme,subjected to washing with hot water, and dehydration at 80° C. underreduced pressure, then diatomaceous earth (1.0 g) was added thereto,stirred, and filtered to remove the enzyme protein.

The resultant product was subjected to first purification step, i.e.molecular distillation, using a centrifugal molecular distillationapparatus at a vacuum pressure of 1.5 Pa, an evaporating surfacetemperature of 230° C. and unreacted free fatty acids and sterols wereremoved as distillate fractions. Then, second purification step wascarried out by adding activated clay in an amount of 10% based on theremaining fraction, stirring at 80° C. under reduced pressure for 30minutes, and removing the activated clay having absorbed the coloringcomponents by filtration. The third purification process, steamdistillation, was carried out using a batch-type steam distillationapparatus under the condition of a vacuum pressure of 500 Pa, adistillation temperature of 150° C., and a distillation time of 1 hour,thereby finally giving a sterol fatty acid ester (56 g) free fromodorous components.

The produced sterol fatty acid esters had a purity of 96.5%, was almosttasteless, odorless and pale yellow in color. The analytic results areshown in Table 2.

EXAMPLE 6

Phytosterols (β-sitosterol content:42.5%) (50 g) and refined rapeseedoil (100 g) were mixed and a suspension in water (50 g) of lipolyticenzyme powder originated from a microorganism of the genus Alcaligenes(90,000 units/g) (1.0 g) was further added thereto, and the syntheticreaction of sterol fatty acid esters were carried out at 40° C. for 24hours with stirring. Then the reaction mixture was heated to 80° C. andstirred at that temperature for 30 min. to inactivate the enzyme,subjected to washing with hot water, and dehydration at 80° C. underreduced pressure, then diatomaceous earth (1.0 g) was added thereto,stirred, and filtered to remove the enzyme protein.

Then first purification step, i.e. molecular distillation treatment,second purification step, i.e. treatment with the adsorbent, and thirdpurification process, i.e. steam distillation treatment were carried outin the same manner as in EXAMPLE 5 and finally sterol fatty acid esters(59 g) were obtained.

The obtained sterol fatty acid esters had a purity of 95.2%, was almosttasteless, odorless and pale yellow in color. The analytic results areshown in Table 2.

EXAMPLE 7

Phytosterols (β-sitosterol content:42.5%) (50 g) and refined olive oil(100 g) were mixed and Vitamin E in an amount of 0.5% and bile salt of2% based on the weight of the mixture were added thereto and dissolvedat 60° C., then a suspension of lipolytic enzyme powder originated froma microorganism of the genus Rhizopus (60,000 units/g) (2.0 g) in water(50 g) was added thereto, and the synthetic reaction of a sterol fattyacid ester was carried out at 60° C. for 24 hours with stirring. Thenthe reaction mixture was heated to 100° C. and stirred at thattemperature for 30 min. to inactivate the enzyme, subjected to washingwith hot water, and dehydration at 80° C. under reduced pressure, thendiatomaceous earth (1.0 g) was added thereto, stirred, and filtered toremove the enzyme protein.

Then first purification step, i.e. molecular distillation treatment,second purification step, i.e. treatment with the adsorbent, and thirdpurification process, i.e. steam distillation treatment, were carriedout in the same manner as in EXAMPLE 5 and finally sterol fatty acidesters (66 g) were obtained.

The obtained sterol fatty acid esters had a purity of 96.4%, was almosttasteless, odorless and pale yellow in color. The analytic results areshown in Table 2.

COMPARATIVE EXAMPLE 2

A synthetic reaction of a sterol fatty acid esters were carried out inthe same manner as in EXAMPLE 5 and subjected to molecular distillationusing a centrifugal molecular distillation apparatus at a vacuumpressure of 1.5 Pa and an evaporating surface temperature of 230° C.,thereby giving sterol fatty acid esters (63 g).

Though the obtained sterol fatty acid ester had a purity of 93.2%, ithad a strong bitterness and a characteristic pungent odor and was brownin color, thereby it had not suitable qualities for a food. The analyticresults are shown in Table 2.

COMPARATIVE EXAMPLE 3

Phytosterols (β-sitosterol content:42.5%) (50 g) and refined soybean oil(100 g) were mixed and lipolytic enzyme powder originated from amicroorganism of the genus Pseudomonas (50,000 units/g) (2.0 g) havingan activity of decomposing sterol fatty acid esters were added and thesynthetic reaction of sterol fatty acid esters were carried out at 40°C. for 24 hours with stirring. Then the reaction mixture was heated to80° C. and stirred at that temperature for 30 min. to inactivate theenzyme, subjected to washing with hot water, and dehydration at 80° C.under reduced pressure, then diatomaceous earth (1.0 g) was addedthereto, stirred, and filtered to remove the enzyme protein.

Then first purification step, i.e. molecular distillation treatment,second purification step, i.e. treatment with the adsorbent, and thirdpurification process, i.e. steam distillation treatment, were carriedout in the same manner as in EXAMPLE 5 and finally sterol fatty acidesters were obtained (86 g).

The obtained sterol fatty acid esters were almost tasteless, odorlessand pale yellow in color, but the purity of the sterol fatty acidesters, 45.7%, were very low. The analytic results are shown in Table 2.TABLE 2 Ex. 5 Ex. 6 Ex. 7 C. Ex. 2 C. Ex. 3 Sterol TLC- 96.5 95.2 96.493.2 45.7 fatty FID % acid esters content Peroxide meq/kg 1.8 1.5 1.93.6 1.5 value (POV) Acid mg-KOH/ 0.1 0.2 0.2 0.9 0.3 value g (AV) ColorGardner 3 3 3 16 3 Scale Odor Sensory almost Almost almost character-almost Evaluation odorless odorless odorless istic odorless strong odorTaste Sensory tasteless tasteless tasteless strong tasteless Evaluationbitter taste

Industrial Applicability

According to the present invention, there is provided an advantage thatdietary sterol fatty acid esters having excellent physiologicalactivities and superior qualities can be produced, by employingphytosterols and fatty acids as starting materials, performing thesynthetic reaction of sterol fatty acid esters using a lipase as acatalyst under strictly controlled conditions, and by purifying thesynthesized sterol fatty acid esters through several steps ofpurification process in order to provide qualities suitable for a food.

Furthermore, according to the present invention, there is provided anadvantage that dietary sterol fatty acid esters having excellentsuperior qualities can be enzymatically produced by employingphytosterols and a fat and oil containing triacylglycerol as the maincomponent, as the starting materials, performing the synthetic reactionof a sterol fatty acid ester using lypolytic enzyme as a catalyst understrictly controlled conditions, and purifying the synthesized sterolfatty acid esters through several steps of purification process in orderto provide qualities suitable for a food.

1. A process for enzymatically producing physiological active dietarysterol fatty acid esters from phytosterols and fatty acids using alipase as a catalyst, wherein fatty acids obtained by enzymatic processor continuous high pressure decomposition process are employed as afatty acid source, a synthetic reaction of sterol fatty acid estersusing a lipase is performed for predetermined time period under theconditions where temperature and water content are controlled,inactivation of the enzyme, dehydration and removal of the enzymeprotein are performed, molecular distillation is carried out to removeunreacted sterols and fatty acids, treatment with an adsorbent iscarried out to remove coloring components, and steam distillation iscarried out to remove odorous components, and thereby sterol fatty acidesters being superior in sensory properties and safety and suitable fora food is obtained.
 2. The process for producing dietary sterol fattyacid esters according to claim 1, wherein the synthetic reaction ofsterol fatty acid esters by lipase is carried out while the watercontent is controlled to be 50% or less based on the amount of thestarting material sterols.
 3. The process for producing dietary sterolfatty acid esters according to claim 1, wherein lipase is a mesophiliclipase, and the synthetic reaction of sterol fatty acid esters areperformed at 30 to 50° C. within 48 hours.
 4. The process for producingdietary sterol fatty acid esters according to claim 1, wherein lipase isa thermostable lipase, and the synthetic reaction of sterol fatty acidesters is performed at 50 to 80° C. within 24 hours, and a substancecapable of inhibiting the inactivation of an enzyme is added when thesynthetic reaction of sterol fatty acid esters are carried out bylipase, and a substance having anti-oxidant action is added when thesynthetic reaction of sterol fatty acid esters are carried out bylipase.
 5. The process for producing dietary sterol fatty acid estersaccording to claim 1, wherein unreacted free sterols and fatty acids areremoved by the molecular distillation performed using a moleculardistillation apparatus at 13.3 Pa or lower and 150 to 250° C. and themolecular distillation treatment being repeated several times.
 6. Theprocess for producing dietary sterol fatty acid esters according toclaim 1, wherein the treatment with an adsorbent is performed using, asthe adsorbent, activated clay in an amount of 1 to 20% based on theweight of the materials to be treated, in the presence of an organicsolvent such as hexane.
 7. The process for producing dietary sterolfatty acid esters according to claim 1, wherein the steam distillationto remove odorous components is carried out at 1330 Pa or lower and at100 to 150° C. to prevent the generation of trans fatty acids.
 8. Theprocess for producing dietary sterol fatty acid esters according toclaim 1, wherein the end product sterol fatty acid esters have sterolfatty acid esters content of 90 wt % or more, the peroxide value of 15or lower, the acid value of 3 or lower and a color scale (Gardner) of 6or lower and is almost odorless as determined by a sensory test.
 9. Theprocess for producing dietary sterol fatty acid esters according toclaim 1, wherein lipase is added in a step-wise manner.
 10. A method forusing dietary sterol fatty acid esters as recited in claim 1 for food inthe form where the sterol fatty acid ester is previously mixed with afat and oil containing triacylglycerol as the main component.
 11. Aprocess for producing dietary sterol fatty acid esters wherein,phytosterols and fats and oils containing triacylglycerols as the maincomponent are employed as starting materials, a synthetic reaction ofsterol fatty acid esters by lipolytic enzyme is carried out for apredetermined time period in a system wherein temperature and watercontent are controlled, molecular distillation, as a first purificationstep is carried out to primarily remove unreacted sterols and fattyacids, treatment with an adsorbent, as a second purification step iscarried out to primarily remove coloring components, steam distillation,as a third purification step is carried out to primarily remove odorouscomponents, thereby a sterol fatty acid ester being superior in sensoryproperties and safety as a food is obtained.
 12. The process forproducing dietary sterol fatty acid esters according to claim 11,wherein the phytosterols used as the starting material containβ-sitosterol in an amount of 20 to 80% by weight.
 13. The process forproducing dietary sterol fatty acid esters according to claim 11,wherein fats and oils containing triacylglycerol as the main componentis one or an admixture of two or more kinds of oils selected fromsoybean oil, rapeseed oil, and olive oil.
 14. The process for producingdietary sterol fatty acid esters according to claim 11, wherein anenzyme having an activity of degrading sterol fatty acid esters areemployed as the lipolytic enzyme.
 15. The process for producing dietarysterol fatty acid esters according to claim 11, wherein an enzyme havingtriacylglycerols decomposition activity is employed as the lipolyticenzyme.
 16. The process for producing dietary sterol fatty acid estersaccording to claim 14 or claim 15, wherein the enzyme havingtriacylglycerol decomposition activity is cholesterol esterase orlipase.
 17. The process for producing dietary sterol fatty acid estersaccording to claim 11, wherein the synthetic reaction of sterol fattyacid esters by lipolytic enzyme is carried out for a predetermined timeperiod in a system wherein temperature and water content are controlled;in which the reaction is carried out at 30 to 60° C. within 48 hourswhile the water content is controlled to be 0.1 to 50% based on theweight of the starting materials.
 18. The process for producing dietarysterol fatty acid esters according to claim 11, wherein the syntheticreaction of sterol fatty acid esters by lipolytic enzyme is carried outfor a predetermined time period in a system wherein temperature andwater content are controlled; in which the reaction is carried out inthe presence of a substance capable of inhibiting the inactivation of anenzyme and a substance having anti-oxidant action, by the use ofthermostable lipolytic enzyme, at 50 to 90° C. within 48 hours while thewater content is controlled to be 0.1 to 50% based on the weight of thestarting materials.
 19. The process for producing dietary sterol fattyacid esters according to claim 18, wherein the synthetic reaction ofsterol fatty acid esters is performed using a lipolytic enzyme which isoriginated from a microorganism of the genus Rhizopus as thethermostable lipolytic enzyme.
 20. The process for producing dietarysterol fatty acid esters according to claim 11, wherein the moleculardistillation as the first purification step to remove primarilyunreacted sterols and fatty acids is performed at 13.3 Pa or lower andat 100 to 250° C.
 21. The process for producing dietary sterol fattyacid esters according to claim 11, wherein the treatment with anadsorbent as the second purification step to remove primarily coloringcomponents is performed at 100° C. or lower using, as the adsorbent,activated clay in an amount of 0.1 to 50% based on the weight of thematerials to be treated.
 22. The process for producing dietary sterolfatty acid esters according to claim 11, wherein the steam distillationas the third purification step is performed to remove odorouscomponents, and by performing it at 1330 Pa or lower and at 50 to 150°C., generation of trans fatty acids is prevented.
 23. The process forproducing dietary sterol fatty acid esters according to claim 11,wherein the end product sterol fatty acid esters has a sterol fatty acidesters content of 90 wt % or more, the peroxide value of 15 or lower,the acid value of 3 or lower and a color scale (Gardner) of 6 or lowerand is almost odorless as determined by a sensory test.